In eukaryotic cells the endoplasmic reticulum (ER) is the primary site of synthesis, folding, and assembly of secreted and integral membrane proteins and their macromolecular complexes (Mu et al., 2008, Cell 134:769-781; Marciniak et al., 2006, Physiol. Rev. 2006:1133-1149; Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529). Maintenance of ER protein homeostasis relies on the timely convergence of multiple pathways that detect homeostatic protein concentration thresholds and control the ebb-and-flow of ER proteins (Mu et al., 2008, Cell 134:769-781; Marciniak et al, 2006, Physiol. Rev. 2006:1133-1149; Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529; Jonikas et al. 2009, Science 323:1693-1697). This process is driven by an intricate network of molecular chaperones and transcription factors. Disruption of ER homeostasis activates stress response pathways including the unfolded protein response (UPR) (Marciniak et al, 2006, Physiol. Rev. 2006:1133-1149; Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529; Kim et al., 2008, Nat. Rev. Drug Discov. 7:1013-1030; Xu et al., 2005, J. Clin. Invest. 2656-2664).
The mammalian UPR comprises at least two phases: an initial alarm phase, followed by a cytoprotective, adaptive phase in which UPR factors are upregulated to enhance the cellular capacity to process increased concentrations of unfolded protein (Marciniak et al, 2006, Physiol. Rev. 2006:1133-1149; Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529; Kim et al., 2008, Nat. Rev. Drug Discov. 7:1013-1030; Xu et al., 2005, J. Clin. Invest. 2656-2664). Imbalanced or altered capacity to respond to ER stress has been implicated in various diseases and disorders (Marciniak et al, 2006, Physiol. Rev. 2006:1133-1149; Kim et al., 2008, Nat. Rev. Drug Discov. 7:1013-1030; Ma et al., 2004, Nat. Rev. Cancer 4:966-977). Protracted ER stress can overwhelm the UPR, leading to autophagy as a secondary survival response (Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529; Bernales et al., 2006, PLoS Biol. 4:e423; Ogata et al., 2006, Mol. Cell Biol. 26:9220-9231; Yorimitsu et al., 2006, J. Biol. Chem. 281:30299-30304). Although the relationship between ER stress, unfolded protein response, and autophagy remains unclear, growing evidence suggests that these responses are likely integrated signaling pathways that modulate cell survival and growth (Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529, He et al., 2009, Annu. Rev. Genet. 43:67-93, Hoyer-Hansen et al., 2007, Cell Death Differ. 14:1576-1582).
Autophagy describes a set of bulk cellular degradation pathways in which large aggregates of misfolded proteins and damaged cellular components, including damaged organelles, are sequestered into membrane bound vesicles called autophagosomes and subsequently targeted for lysosomal degradation (He et al., 2009, Annu. Rev. Genet. 43:67-93; Levine et al., 2004, Dev. Cell 6:463-477). Complete autophagy comprises autophagosome fusion with lysosomes to form autolysosomes, wherein the sequestered proteins and lipids are subsequently degraded by autophagic degradation or flux (He et al., 2009, Annu. Rev. Genet. 43:67-93; Levine et al., 2004, Dev. Cell 6:463-477). Autophagy occurs under basal conditions in many tissues and is involved in cellular differentiation and development. It is also activated or hyperactivated in conditions of nutrient starvation and cellular stress (Levine et al., 2004, Dev. Cell 6:463-477, Mizushima et al., 2008, Nature 451:1069-1075), to maintain energy levels and to sequester and remove damaged and cytotoxic cellular components (Levine et al., 2004, Dev. Cell 6:463-477; Mizushima et al., 2008, Nature 451:1069-1075). Thus, autophagy plays important roles in cellular homeostasis and disease prevention, and defective autophagy has been implicated in neurodegenerative disease and cancer (Levine et al., 2008, Cell 132:27-42; Mizushima et al., 2008, Nature 451:1069-1075; White et al., 2009, Clin. Cancer Res. 15:5308-5316).
Autophagy has been shown to influence tumor cell growth and tumorigenesis (Levine et al., 2008, Cell 132:27-42; White et al., 2009, Clin. Cancer Res. 15:5308-5316; Degenhardt et al., 2006, Cancer Cell 10:304-312; Mathew et al., 2007, Nat. Rev. Cancer 7:961-967). Autophagy may serve a cytoprotective role in cancer cells (Levine et al., 2008, Cell 132:27-42; Mizushima et al., 2008, Nature 451:1069-1075; White et al., 2009, Clin. Cancer Res. 15:5308-5316; Degenhardt et al., 2006, Cancer Cell 10:304-312). Several antineoplastic agents have been shown to induce autophagy (Rubinsztein et al., 2007, Rev. Drug Discov. 6:304-312). However, in many cases it remains unclear whether cell death occurs by autophagy, whether cell death is associated with autophagy, or whether autophagy is a survival response to cytotoxic chemotherapy (Levine et al., 2004, Dev. Cell 6:463-477; Levine et al., 2008, Cell 132:27-42; White et al., 2009, Clin. Cancer Res. 15:5308-5316; Hippert et al., 2006, Cancer Res. 66:9349-9351). Emerging data suggest that autophagy participates in integrated responses to cellular stress that determine cell death versus survival. The proteins and pathways that regulate these integrated stress responses are just beginning to be defined (Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529; Kim et al., 2008, Nat. Rev. Drug Discov. 7:1013-1030, Levine et al., 2004, Dev. Cell 6:463-477; Rubinsztein et al., 2006, Neuron 54:9349-9351).
Sigma receptors, first proposed 30 years ago (Martin et al., 1976, J. Pharmacol. Exp. Ther. 197:517-532), are distinct from classical opioid receptors (Su, 1982, J. Pharmacol. Exp. Ther. 223:284-290). Binding studies suggest at least two Sigma receptor subtypes, of which only the Sigma1 receptor (hereinafter “Sigma1”) has been cloned, whereas the identity of Sigma2 remains unclear (Hanner et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:8072-8077; Vilner et al., 1995, Cancer Res. 55:408-413). Sigma1 is highly conserved among mammals (greater than 80% amino acid identity), but shares no significant homology with any traditional receptor family or other mammalian protein (White et al., 2009, Clin. Cancer Res. 15:5308-5316; Mathew, et al., 2007, Nat. Rev. Cancer 7:961-967). Cloned Sigma1 is a 26 kilodalton integral membrane protein (Hanner et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:8072-8077; Pal et al., 2007, Mol. Pharmacol. 72:921-933; Aydar et al., 2007, Neuron 34:399-410; Hayashi et al., 2007, Cell 131:596-610). It is found primarily in the ER, and can translocate to the plasma membrane, other organelles, and endoplasmic membrane microdomains (Hanner et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:8072-8077; Aydar et al., 2007, Neuron 34:399-410; Hayashi et al., 2007, Cell 131:596-610; Hayashi et al., 2003, J. Pharmacol. Exp. Ther. 306:718-725; Palmer et al., 2007, Cancer Res. 67:11166-11175). Sigma receptors are highly expressed in tumor cell lines, including prostate and breast adenocarcinoma (Vilner et al., 1995, Cancer Res. 55:408-413; Berthosis et al., 2003, Br. J. Cancer 88:438-446; Piergentili et al., J. Med. Chem. 53:1261-1269). Some Sigma ligands are reported as antitumor agents (Berthosis et al., 2003, Br. J. Cancer 88:438-446; Vilner et al., 1995, J. Neurosci. 15:117-134). Interestingly, putative Sigma antagonists, but not agonists, inhibit prostate carcinoma proliferation in vitro and inhibit tumor growth in tumor xenograft experiments (Berthosis et al., 2003, Br. J. Cancer 88:438-446; Spruce et al., 2004, Cancer Res. 64:4875-4886). Recent work has described Sigma ligand-induced cell death by lysosomal destabilization and oxidative stress.
There are numerous examples of clinically used compounds that bind Sigma1 with high affinity and thus are considered Sigma1 ligands, such as haloperidol, a widely used antipsychotic that also binds D2 receptors with similar affinity and whose anti-psychotic properties are primarily understood as D2 mediated (Seeman, et al., 1975, Science 188:1217-1219; Seeman et al., 1976, Nature 261:717-719), and the hallucinogen N,N-dimethyltryptamine, hypothesized to be an endogenous Sigma1 regulator (Fontanilla et al., 2009, Science 323:934-937). Sigma receptors have proved to be highly attractive pharmacological targets for the treatment of various pathologies, such as neuropathic pain (de la Puente et al., 2009, Pain 145:294-303), depression (Skuza, 2003, Pol. J. Pharmacol. 55:923-934), cocaine abuse (Matsumoto et al., 2003, Eur. J. Pharmacol. 469:1-12), epilepsy (Lin et al., 1997, Med. Res. Rev. 17:537-572), psychosis (Rowley et al., 2001, J. Med. Chem. 44:477-501), and Alzheimer's and Parkinson's disease (Maurice et al., 1997, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 21:69-102; Marrazzo et al., 2005, NeuroReport 16:1223-1226). Recent reports demonstrate a genetic link between the Sigma1 receptor gene (SIGMAR1) and Amyotrophic lateral sclerosis (ALS) (Al-Saif et al., 2011, Ann Neurol. 70(6):913-9), as well as Frontotemporal Lobar Degeneration (FTLD) (Luty et al., 2010, Ann Neurol. 2010 68(5):639-49). Moreover, Sigma1 antagonists and Sigma2 agonists may be useful as anticancer agents and selective tumor imaging agents (Akhter et al., 2008, Nucl. Med. Biol. 35:29-34; Tu et al., 2007, J. Med. Chem. 50:3194-3204).
Sigma1 can function as a molecular chaperone at the ER-mitochondrion interface at least in certain model cell lines (Hayashi & Su, 2007, Cell 131(3):596-610). However, the physiological role of Sigma receptors as well as their role in neurodegenerative disease and cancer remains unclear. In vitro, treatment with a Sigma antagonist results in apoptotic cell death following prolonged treatment, with Sigma ligand time-action and dose-response, depending on the Sigma antagonist and cell line (Berthosis et al., 2003, Br. J. Cancer 88:438-446; Piergentili et al., J. Med. Chem. 53:1261-1269; Spruce et al., 2004, Cancer Res. 64:4875-4886; Vilner et al., 1995, J. Neurosci. 15:117-134). Yet, a mechanistic understanding of the Sigma1 receptor system remains elusive.
Most prostate cancer patients become unresponsive to initially effective hormone- and chemotherapy as prostate tumor cells eventually adapt and develop resistance. Treatment with Sigma antagonists leads to apoptotic cell death of both androgen-sensitive and androgen-insensitive prostate cancer cells (Berthosis et al., 2003, Br. J. Cancer 88:438-446; Spruce et al., 2004, Cancer Res. 64:4875-4886). Although some insight has been gained into how prostate cancer cells develop such resistance, currently there are few alternatives to treat hormone refractory (castration resistant) prostate cancer. Emerging therapies to treat intractable, advanced prostate cancers target protein processing and chaperone pathways that maintain prostate tumor growth and survival.
There is a need in the art to identify compounds useful in the treatment of intractable, advanced cancers. Such compounds may target protein processing, protein synthesis, protein folding, protein transport, protein localization, protein assembly into functional macromolecular complexes, and related chaperone pathways, all of which may help maintain tumor growth, survival and metastasis. The present invention addresses this unmet need.